Masashi Mukohda

Omentin, a novel adipokine, induces vasodilation in rat isolated blood vessels.

Omentin is a recently identified adipose tissue-derived cytokine and is implicated in obesity-related cardiovascular disorders. In the present study, we tested the hypothesis that omentin could directly affect vascular reactivity of isolated blood vessels. In endothelium-intact rat isolated aorta, pretreatment with omentin (300 ng/ml, 30 min) inhibited noradrenaline (NA; 1 nM-1 microM)-induced concentration-dependent contraction. In NA (100 nM)-pre-contracted aorta, omentin (1-300 ng/ml) directly induced an endothelium-dependent relaxation. While a nitric oxide (NO) synthase (NOS) inhibitor, N(G)-nitro-l-arginine methyl ester (100 microM, 30 min) inhibited the relaxation, a PI3K/Akt inhibitor, LY294002 (10 microM, 30 min) or a tyrosine kinase inhibitor, genistein (30 microM, 30 min) was ineffective. Omentin (300 ng/ml, 5 min) induced a phosphorylation of endothelial NOS at serine 1177 but not a phosphorylation of Akt at serine 473. Omentin (1-300 ng/ml) also relaxed NA pre-contracted mesenteric artery. Present study for the first time demonstrated that omentin has a vasodilating effect on isolated blood vessels, which is mediated through endothelium-derived NO.

A novel adipocytokine, nesfatin-1 modulates peripheral arterial contractility and blood pressure in rats

Nesfatin-1 is a novel adipocytokine which exerts not only anorexigenic but also hypertensive roles through acting on hypothalamus melanocortin-3/4 receptors. Although it is logical to hypothesize that nesfatin-1 could also affect the contractile reactivity of peripheral blood vessels, it still remains to be examined. The present study was performed to test the hypothesis. In both endothelium-intact and -denuded mesenteric artery of rats, acute treatment with nesfatin-1 (10nM, 30min pretreatment) had no influence on the noradrenaline- and 5-hydroxytryptamine-induced concentration-dependent contractions. Chronic treatment of mesenteric artery with nesfatin-1 (10nM, 3days) using organ-culture method had also no influence on the agonists-induced contractions. In contrast, nesfatin-1 (10nM, 30min) significantly inhibited the sodium nitroprusside (SNP)-induced relaxations of smooth muscle in mesenteric artery. A membrane permeable cyclic GMP (cGMP) analog, 8-bromo-cGMP-induced relaxations were not affected by nesfatin-1. Consistently, the SNP-induced cGMP production in smooth muscle was impaired by nesfatin-1. Intravenous application of nesfatin-1 to rats not only increased blood pressure but also impaired the SNP-induced decreases in blood pressure. The present study for the first time reveals that nesfatin-1 affects peripheral arterial blood vessel and inhibits the nitric oxide donor-induced smooth muscle relaxations via impairing the cGMP production. The results are the first to demonstrate that nesfatin-1 modulates blood pressure through directly acting on peripheral arterial resistance.

Telmisartan inhibits methylglyoxal-mediated cell death in human vascular endothelium

Methylglyoxal (MGO) is a metabolite of glucose. Since serum MGO level is increased in diabetic patients, MGO is implicated in diabetic complications related to vascular injury. We have recently demonstrated that glucose metabolite is a more powerful stimulant for endothelial cells (ECs) injury rather than glucose or advanced glycation-end products. Recent clinical trials suggest that angiotensin receptor blockers are effective to prevent diabetes-associated cardiovascular disorders beyond blood pressure lowering effect. To explore the mechanisms, we examined effects of telmisartan on MGO-induced ECs injury. Treatment of human umbilical vein ECs with MGO (560 microM) induced time-dependent (0-24 h) cell death. MGO-induced cell death was apoptosis since MGO increased cleaved caspase-3 expression. Telmisartan (0.1-10 microM) inhibited MGO-induced cell death and caspase-3 activation. These results indicate that telmisartan prevents MGO-induced apoptosis by inhibiting caspase-3 activation, which might explain at least in part the beneficial effects of telimisartan against diabetes-related cardiovascular diseases.

Long-term methylglyoxal treatment impairs smooth muscle contractility in organ-cultured rat mesenteric artery

Methylglyoxal (MGO), a metabolite of glucose accumulates in vascular tissues of hypertensive rats. We recently showed that short-term (30min) treatment with MGO inhibits noradrenaline (NA)-induced smooth muscle contraction in rat aorta and mesenteric artery. In the present study, long-term effect of MGO was examined using organ culture method. The contractility, morphology, and protein expression of rat mesenteric artery after organ culture with MGO for 3 days were examined. MGO (4 and 42μM) inhibited NA (0.1nM to 3μM) or KCl (72.7mM)-induced contraction. The inhibitory effect was higher in endothelium-denuded than endothelium-intact artery. An anti-oxidant drug, N-acetyl-l-cysteine (NAC; 1mM) or an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), gp91ds-tat (0.1μM) prevented the inhibitory effect of MGO. MGO increased superoxide production as detected by lucigenin assay. In the medial layer of the arteries cultured with MGO, apoptotic morphological change was observed, and NAC or gp91ds-tat prevented it. MGO significantly increased expression of a homolog of gp91(phox), NOX1 but not gp91(phox) as determined by Western blotting. An NF-κB inhibitor, pyrrolidine dithiocarbamate prevented the MGO-induced NOX1 expression. MGO had no effect on protein expression of p22(phox), p67(phox), p47(phox), as well as superoxide dismutase (SOD)-1, SOD-2 and SOD-3. Present results indicate that long-term MGO treatment has an inhibitory effect on contractility of isolated blood vessel, which is likely mediated via increased NOX1-derived superoxide production and subsequent apoptosis.

Interference with PPARγ in endothelium accelerates angiotensin II-induced endothelial dysfunction

The ligand activated nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) in the endothelium regulates vascular function and blood pressure (BP). We previously reported that transgenic mice (E-V290M) with selectively targeted endothelial-specific expression of dominant negative PPARγ exhibited endothelial dysfunction when treated with a high-fat diet, and exhibited an augmented pressor response to angiotensin II (ANG II). We hypothesize that interference with endothelial PPARγ would exacerbate ANG II-induced endothelial dysfunction. Endothelial function was examined in E-V290M mice infused with a subpressor dose of ANG II (120 ng·kg(-1)·min(-1)) or saline for 2 wk. ANG II infusion significantly impaired the responses to the endothelium-dependent agonist acetylcholine both in basilar and carotid arteries from E-V290M but not NT mice. This impairment was not due to increased BP, which was not significantly different in ANG II-infused E-V290M compared with NT mice. Superoxide levels, and expression of the pro-oxidant Nox2 gene was elevated, whereas expression of the anti-oxidant genes Catalase and SOD3 decreased in carotid arteries from ANG II-infused E-V290M mice. Increased p65 and decreased Iκ-Bα suggesting increased NF-κB activity was also observed in aorta from ANG II-infused E-V290M mice. The responses to acetylcholine were significantly improved both in basilar and carotid arteries after treatment with Tempol (1 mmol/l), a scavenger of superoxide. These findings provide evidence that interference with endothelial PPARγ accelerates ANG II-mediated endothelial dysfunction both in cerebral and conduit arteries through an oxidative stress-dependent mechanism, suggesting a role for endothelial PPARγ in protecting against ANG II-induced endothelial dysfunction.

PPARγ Regulation in Hypertension and Metabolic Syndrome

Dysregulation of peroxisome proliferator-activated receptor gamma (PPARγ) activity leads to significant alterations in cardiovascular and metabolic regulation. This is most keenly observed by the metabolic syndrome-like phenotypes exhibited by patients carrying mutations in PPARγ. We will summarize recent findings regarding mechanisms of PPARγ regulation in the cardiovascular and nervous systems focusing largely on PPARγ in the smooth muscle, endothelium, and brain. Canonically, PPARγ exerts its effects by regulating the expression of target genes in these cells, and we will discuss mechanisms by which PPARγ targets in the vasculature regulate cardiovascular function. We will also discuss emerging evidence that PPARγ in the brain is a mediator of appetite and obesity. Finally, we will briefly review how novel PPARγ activators control posttranslational modifications of PPARγ and their prospects to offer new therapeutic options for treatment of metabolic diseases without the adverse side effects of thiazolidinediones which strongly activate transcriptional activity of PPARγ.

Endothelial PPAR-γ provides vascular protection from IL-1β-induced oxidative stress

Loss of peroxisome proliferator-activated receptor (PPAR)-γ function in the vascular endothelium enhances atherosclerosis and NF-κB target gene expression in high-fat diet-fed apolipoprotein E-deficient mice. The mechanisms by which endothelial PPAR-γ regulates inflammatory responses and protects against atherosclerosis remain unclear. To assess functional interactions between PPAR-γ and inflammation, we used a model of IL-1β-induced aortic dysfunction in transgenic mice with endothelium-specific overexpression of either wild-type (E-WT) or dominant negative PPAR-γ (E-V290M). IL-1β dose dependently decreased IκB-α, increased phospho-p65, and increased luciferase activity in the aorta of NF-κB-LUC transgenic mice. IL-1β also dose dependently reduced endothelial-dependent relaxation by ACh. The loss of ACh responsiveness was partially improved by pretreatment of the vessels with the PPAR-γ agonist rosiglitazone or in E-WT. Conversely, IL-1β-induced endothelial dysfunction was worsened in the aorta from E-V290M mice. Although IL-1β increased the expression of NF-κB target genes, NF-κB p65 inhibitor did not alleviate endothelial dysfunction induced by IL-1β. Tempol, a SOD mimetic, partially restored ACh responsiveness in the IL-1β-treated aorta. Notably, tempol only modestly improved protection in the E-WT aorta but had an increased protective effect in the E-V290M aorta compared with the aorta from nontransgenic mice, suggesting that PPAR-γ-mediated protection involves antioxidant effects. IL-1β increased ROS and decreased the phospho-endothelial nitric oxide synthase (Ser(1177))-to-endothelial nitric oxide synthase ratio in the nontransgenic aorta. These effects were completely abolished in the aorta with endothelial overexpression of WT PPAR-γ but were worsened in the aorta with E-V290M even in the absence of IL-1β. We conclude that PPAR-γ protects against IL-1β-mediated endothelial dysfunction through a reduction of oxidative stress responses but not by blunting IL-1β-mediated NF-κB activity.

Effect of selective expression of dominant-negative PPARγ in pro-opiomelanocortin neurons on the control of energy balance

Peroxisome proliferator-activated receptor-γ (PPARγ), a master regulator of adipogenesis, was recently shown to affect energy homeostasis through its actions in the brain. Deletion of PPARγ in mouse brain, and specifically in the pro-opiomelanocortin (POMC) neurons, results in resistance to diet-induced obesity. To study the mechanisms by which PPARγ in POMC neurons controls energy balance, we constructed a Cre-recombinase-dependent conditionally activatable transgene expressing either wild-type (WT) or dominant-negative (P467L) PPARγ and the tdTomato reporter. Inducible expression of both forms of PPARγ was validated in cells in culture, in liver of mice infected with an adenovirus expressing Cre-recombinase (AdCre), and in the brain of mice expressing Cre-recombinase either in all neurons (NES(Cre)/PPARγ-P467L) or selectively in POMC neurons (POMC(Cre)/PPARγ-P467L). Whereas POMC(Cre)/PPARγ-P467L mice exhibited a normal pattern of weight gain when fed 60% high-fat diet, they exhibited increased weight gain and fat mass accumulation in response to a 10% fat isocaloric-matched control diet. POMC(Cre)/PPARγ-P467L mice were leptin sensitive on control diet but became leptin resistant when fed 60% high-fat diet. There was no difference in body weight between POMC(Cre)/PPARγ-WT mice and controls in response to 60% high-fat diet. However, POMC(Cre)/PPARγ-WT, but not POMC(Cre)/PPARγ-P467L, mice increased body weight in response to rosiglitazone, a PPARγ agonist. These observations support the concept that alterations in PPARγ-driven mechanisms in POMC neurons can play a role in the regulation of metabolic homeostasis under certain dietary conditions.

Hypertension-Causing Mutation in Peroxisome Proliferator–Activated Receptor γ Impairs Nuclear Export of Nuclear Factor-κB p65 in Vascular Smooth Muscle

Selective expression of dominant negative (DN) peroxisome proliferator–activated receptor &ggr; (PPAR&ggr;) in vascular smooth muscle cells (SMC) results in hypertension, atherosclerosis, and increased nuclear factor-&kgr;B (NF-&kgr;B) target gene expression. Mesenteric SMC were cultured from mice designed to conditionally express wild-type (WT) or DN-PPAR&ggr; in response to Cre recombinase to determine how SMC PPAR&ggr; regulates expression of NF-&kgr;B target inflammatory genes. SMC-specific overexpression of WT-PPAR&ggr; or agonist-induced activation of endogenous PPAR&ggr; blunted tumor necrosis factor &agr; (TNF-&agr;)–induced NF-&kgr;B target gene expression and activity of an NF-&kgr;B–responsive promoter. TNF-&agr;–induced gene expression responses were enhanced by DN-PPAR&ggr; in SMC. Although expression of NF-&kgr;B p65 was unchanged, nuclear export of p65 was accelerated by WT-PPAR&ggr; and prevented by DN-PPAR&ggr; in SMC. Leptomycin B, a nuclear export inhibitor, blocked p65 nuclear export and inhibited the anti-inflammatory action of PPAR&ggr;. Consistent with a role in facilitating p65 nuclear export, WT-PPAR&ggr; coimmunoprecipitated with p65, and WT-PPAR&ggr; was also exported from the nucleus after TNF-&agr; treatment. Conversely, DN-PPAR&ggr; does not bind to p65 and was retained in the nucleus after TNF-&agr; treatment. Transgenic mice expressing WT-PPAR&ggr; or DN-PPAR&ggr; specifically in SMC (S-WT or S-DN) were bred with mice expressing luciferase controlled by an NF-&kgr;B–responsive promoter to assess effects on NF-&kgr;B activity in whole tissue. TNF-&agr;–induced NF-&kgr;B activity was decreased in aorta and carotid artery from S-WT but was increased in vessels from S-DN mice. We conclude that SMC PPAR&ggr; blunts expression of proinflammatory genes by inhibition of NF-&kgr;B activity through a mechanism promoting nuclear export of p65, which is abolished by DN mutation in PPAR&ggr;.